PROJECT SUMMARY/ABSTRACT In a multicellular organism, control of cell proliferation and differentiation is determined by the net output of multiple pathways. This is illustrated by cooperation between the well-known Rb tumor suppressor pathway and the emerging Hippo tumor suppressor pathway. However, these types of studies are intrinsically complex and therefore model organisms such as Drosophila are attractive alternatives to complement analogous studies in mammalian systems. This is due primarily to the relative simplicity yet high conservation of the Drosophila Rb and Hippo pathways. In the previous cycle of this grant, we explored the mechanistic details of cooperation between the Hippo and Rb pathways in the regulation of cell proliferation and identified new roles of these pathways. We discovered a novel function of the Rb pathway in the regulation of expression of mitochondria-associated genes and showed that the loss of such regulation results in severe mitochondrial defects, both in flies and in human tumor cells. We also showed that the Rb pathway has an essential function in Drosophila adult skeletal muscles that is necessary and sufficient for animal viability. Our proposal builds on these observations to investigate these new aspects of the Rb and Hippo pathways beyond their conventional role in cell proliferation. In particular, we will determine how the two pathways cooperate to regulate the mitochondrial function in flies and will use this knowledge to guide us towards improving the therapeutic effects of a CDK4/6 inhibitor in tumor cells. Additionally, we will explore the importance of the Rb pathway in adult skeletal muscles and define the underlying mechanisms. Finally, we will use a novel genetic cell-labeling system, single-cell approaches and advanced genomic tools to elucidate the molecular mechanisms by which the Hippo and Rb pathways maintain terminal differentiation in vivo. Collectively, these studies will enhance our knowledge of the cooperation of tumor suppressor pathways and will contribute to the identification of new therapeutic targets.